水稻OsPAL6与OsPetc蛋白互作响应纹枯菌侵染的机制研究

doi:10.16819/j.1001-7216.2026.251203

摘要/Abstract

摘要：

【目的】由立枯丝核菌引起的水稻纹枯病是世界上最严重的水稻病害之一，严重影响水稻产量。研究水稻苯丙氨酸解氨酶编码基因OsPAL6在水稻-立枯丝核菌互作中发挥的功能，同时探究OsPAL6和OsPetc可能参与的抗病通路具有重要意义。【方法】利用抗感水稻品种接种立枯丝核菌后的转录组数据筛选到OsPAL6，并通过qPCR验证OsPAL6对纹枯病菌侵染的响应以及OsPAL6在不同组织中的相对表达量。构建OsPAL6的亚细胞定位载体，检测OsPAL6在烟草细胞中的定位。构建OsPAL6的转基因株系，对不同株系离体叶片接种纹枯病菌，测量相对病斑面积。此外，通过酵母双杂筛库，筛选到与OsPAL6可能的互作蛋白OsPetc，并用酵母双杂交(Y2H)，双分子荧光互补(BiFC)实验验证二者是否互作。【结果】OsPAL6在不同时间段均响应纹枯病菌侵染，且在叶片中的相对表达量最高。OsPAL6定位在细胞核和细胞质膜，过表达植株的相对病斑面积显著低于野生型植株，突变体植株的相对病斑面积显著高于野生型植株，且OsPAL6和OsPetc相互作用。【结论】OsPAL6正调控水稻对纹枯病的抗性，且可能通过与OsPetc互作而发挥抗病功能。综上，本研究揭示了苯丙氨酸代谢通路参与水稻抗纹枯病过程的调控网络，为水稻-立枯丝核菌互作机制研究提供新思路。

关键词: 水稻, 立枯丝核菌, OsPAL6, OsPetc, 双分子荧光互补

Abstract:

【Objective】 Rice sheath blight, caused by Rhizoctonia solani, is one of the most devastating rice diseases worldwide, significantly impacting rice yield. Investigating the function of rice phenylalanine ammonia-lyase encoding gene OsPAL6 in the rice-Rhizoctonia solani interaction and exploring the disease resistance pathways potentially involving OsPAL6 and OsPetc is of great importance. 【Method】 OsPAL6 was initially identified based on previous transcriptome data of resistant and susceptible rice varieties inoculated with Rhizoctonia solani. Subsequently, RT-qPCR was performed to verify whether OsPAL6 responds to sheath blight pathogen infection and to determine its relative expression levels in different tissues. A subcellular localization vector for OsPAL6 was constructed to determine its localization in tobacco cells. OsPAL6-overexpressing transgenic rice lines were generated, and detached leaves from different lines were inoculated with Rhizoctonia solani to measure the relative lesion area. Furthermore, a yeast two-hybrid library screen was performed to identify potential interacting proteins of OsPAL6. The interaction between OsPAL6 and OsPetc was confirmed using yeast two-hybrid(Y2H) and bimolecular fluorescence complementation (BiFC) assays. 【Result】 OsPAL6 responded to Rhizoctonia solani infection at all tested time points, with the highest relative expression level observed in leaves. Subcellular localization analysis revealed that OsPAL6 was localized in both the nucleus and the cytoplasmic membrane. Overexpression lines exhibited a significantly smaller relative lesion area compared to wild-type plants, whereas mutant plants showed a significantly larger relative lesion area. Furthermore, an interaction between OsPAL6 and OsPetc was demonstrated. 【Conclusion】 OsPAL6 positively regulates rice resistance to sheath blight. This function is likely mediated through its interaction with OsPetc. In summary, this study reveals the regulatory network involving the phenylalanine metabolic pathway in the defense against sheath blight and provides new insights into the interaction mechanism between rice and Rhizoctonia solani.

Key words: rice, Rhizoctonia solani, OsPAL6, OsPetc, bimolecular fluorescence complementation

魏松红, 王奕鸣, 罗杭, 张良坤, 王梦雨, 王妍. 水稻OsPAL6与OsPetc蛋白互作响应纹枯菌侵染的机制研究[J]. 中国水稻科学, 2026, 40(6): 767-773.

WEI Songhong, WANG Yiming, LUO Hang, ZHANG Liangkun, WANG Mengyu, WANG Yan. Molecular Mechanism of OsPAL6-OsPetc Interaction in Rice Response to Rhizoctonia solani Infection[J]. Chinese Journal OF Rice Science, 2026, 40(6): 767-773.

图/表 5

表1 本研究中使用的引物

Table 1. Primers used in this study

引物 Primer		引物序列 Primer sequence (5’-3’)	引物用途 Usage
OsPAL6-QPCR	Primer-F	TACTCTGGCATCCGGTTCGAG	组织表达分析，相对表达量检测 Tissue expression analysis, relative expression level detection
OsPAL6-QPCR	Primer-R	GATGTAGGAGAGCGGGACGA
OsUBC-QPCR	Primer-F	CCGTTTGTAGAGCCATAATTGCA	内参基因 Reference gene
OsUBC-QPCR	Primer-R	AGGTTGCCTGAGTCACAGTTAAGTG	内参基因 Reference gene
pGBKT7-OsPAL6-CDS	Primer-F	TGCATATGGCCATGGAGGCCGAATTCATGGAG TGCGAGAACGGG	酵母双杂交 Yeast two-hybrid
pGBKT7-OsPAL6-CDS	Primer-R	TTATGCTAGTTATGCGGCCGCTGCAGTCAGCAG ATGGGCAGAGGCT
pGADT7-OsPetc-CDS	Primer-F	ATATGGCCATGGAGGCCAGTGAATTCATGGCCTC CACCGCGCT
pGADT7-OsPetc-CDS	Primer-R	ATCTGCAGCTCGAGCTCGATGGATCCTTACGCCC ACCACGGGTTG
pCAMBIA1302-OsPAL6-CDS	Primer-F	AGAACACGGGGGACTCTTGACCATGGATGGAGT GCGAGAACGGG	亚细胞定位 Subcellular localization
pCAMBIA1302-OsPAL6-CDS	Primer-R	GTGAAAAGTTCTTCTCCTTTACTAGTTCAGCAGA TGGGCAGAGGCT
pCAMBIA1302-OsPetc-CDS	Primer-F	AGAACACGGGGGACTCTTGACCATGGATGGCCT CCACCGCGCT
pCAMBIA1302-OsPetc-CDS	Primer-R	GTGAAAAGTTCTTCTCCTTTACTAGTTTACGCCC ACCACGGGTTG
OsPAL6-PXC-CDS	Primer-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAT GGAGTGCGAGAACGGGCA	烟草双分子荧光互补 Bimolecular fluorescence complementation in tobacco
OsPAL6-PXC-CDS	Primer-R	GGGGACCACTTTGTACAAGAAAGCTGGGTGGCA GATGGGCAGAGGCTC
OsPetc-PXN-CDS	Primer-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAT GGCCTCCACCGCGCTCT
OsPetc-PXN-CDS	Primer-R	GGGGACCACTTTGTACAAGAAAGCTGGGTGCGC CCACCACGGGTTGTC
pRTVcVN-OsPAL6	Primer-F	CAGATTACGCTGGATCCATGGAGTGCGAGAACG GGCA	水稻双分子荧光互补 Bimolecular fluorescence complementation in rice
pRTVcVN-OsPAL6	Primer-R	GCCGCACTAGTAAGCTTTCAGCAGATGGGCAGAG
pRTVcVC-OsPetc	Primer-F	CAGATCCAGTGGGATCCATGGCCTCCACCGCG
pRTVcVC-OsPetc	Primer-R	GCCGCACTAGTAAGCTTCGCCCACCACGGGTTGT

图1 OsPAL6的筛选和生物信息学分析 A：热图表示水稻4个PAL家族成员在转录组数据中的表达水平。B：OsPAL6的系统进化树，使用MEGA11软件构建。C：水稻叶片不同接种时间点OsPAL6的相对表达量，数据表示平均值±标准误（n=3）。D: 水稻PAL家族成员的多序列比对，使用Jalview软件构建。***，****表示通过单因素方差分析中Dunnett检验确定的显著性差异；***, P<0.001，****, P<0.0001。

Fig. 1. Screening and bioinformatics analysis of the OsPAL6 gene A, Heat map represents the expression levels of four PAL family members in rice based on transcriptomic data; B, Phylogenetic tree of OsPAL6 constructed using MEGA11 software; C, Relative expression levels of OsPAL6 in rice leaves at different time points following inoculation. Data are presented as mean±standard error (n=3); D, Multiple sequence alignment of rice PAL family members conducted with Jalview software; Data are presented as mean ± standard error (n=3). *** and **** indicate significant differences determined by the Dunnett's test in one-way ANOVA；***, P< 0.001 and ****, P< 0.0001.

图2 OsPAL6的组织表达分析和亚细胞定位 A：OsPAL6在不同组织中的相对表达量，数据表示平均值±标准误（n=3）。B：OsPAL6在烟草细胞中的亚细胞定位，以pCAMBIA1302空质粒为对照，比例尺=50 μm。不同字母表示通过单因素方差分析中Tukey检验确定差异显著(P<0.05)。

Fig. 2. Tissue-specific expression analysis and subcellular localization of OsPAL6 A, Relative expression levels of OsPAL6 in different tissues. Data represent mean ± SE (n=3). B, Subcellular localization of OsPAL6 in tobacco cells, using the pCAMBIA1302 plasmid as a control, bar=50 μm. Different letters indicate significant differences determined by the Tukey's test in one-way ANOVA(P < 0.05).

图3 OsPAL6的抗病功能研究 A：ospal6的突变位点分析，采用CRISPR/Cas9方法敲除OsPAL6，敲除位点位于第一个外显子，缺失2 bp碱基。B：OsPAL6在野生型和过表达植株中的相对表达量，数据表示平均值±标准误（n=3）。C：OsPAL6正调控水稻对纹枯病的抗性。D：三种类型植株接种纹枯病菌后叶片的相对病斑面积，数据表示平均值±标准误（n=3）。**表示通过单尾t检验确定的差异显著；****表示通过单因素方差分析中Dunnett检验差异显著；**P<0.01，****P<0.0001。

Fig. 3. Research on the disease resistance function of OsPAL6 A, Analysis of mutation sites in ospal6. OsPAL6 was knocked out using the CRISPR/Cas9 method, with the knockout site located in the first exon, resulting in a 2-bp deletion. B, Relative expression levels of OsPAL6 in the wild-type and the overexpression plants. Data represent mean ± SE (n=3). C, OsPAL6 positively regulates rice resistance to sheath blight. D, Relative lesion area on leaves from the three types of plants after inoculation with Rhizoctonia solani. Data represent mean ± SE (n=3). ** indicates a significant difference determined by a one-tailed t-test; **** indicates significant differences determined by Dunnett's test in one-way ANOVA; **P < 0.01, ****P < 0.0001.

图4 OsPAL6与OsPetc的相互作用 A：酵母双杂交实验证明OsPAL6与OsPetc互作。B：烟草双分子荧光互补（BiFC）实验证明OsPAL6和OsPetc在细胞核中互作。C：水稻双分子荧光互补（BiFC）实验证明OsPAL6和OsPetc在细胞核中互作。

Fig. 4. Interaction between OsPAL6 and OsPetc A, Yeast two-hybrid assay demonstrates the interaction between OsPAL6 and OsPetc. B, The tobacco Bimolecular fluorescence complementation (BiFC) assay confirms that OsPAL6 interacts with OsPetc in cell nucleus; C, The Rice Bimolecular fluorescence complementation (BiFC) assay confirms that OsPAL6 interacts with OsPetc in the cell nucleus.

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